RESUMEN
Triazoles are the only compounds used as antibiotics in both medicine and agriculture. The presence of triazoles in the environment can contribute to the acquisition of azole resistance among isolates of Aspergillus fumigatus. The objective of this study was to investigate the effect of A. fumigatus exposure to triazoles on susceptibility to these compounds. Seventeen triazole-resistant and 21 triazole-sensitive A. fumigatus isolates were examined. The isolates were transferred 20 times on the Sabouraud medium supplemented with posaconazole, itraconazole or voriconazole, followed by five times on the medium not supplemented. The minimum inhibitory concentrations of antimycotics were examined according to the EUCAST broth microdilution method after the 20th transfer and also the 25th transfer. In addition, the expression levels of genes mdr1, mdr2, mdr3, atrF, cyp51A and cyp51B were determined. Cultivation of A. fumigatus on media supplemented with posaconazole, itraconazole and voriconazole resulted in the acquisition of resistance to the tested triazoles of all examined isolates. After recultivation on Sabouraud without azoles, most of the isolates lost their acquired resistance. The long-term use of triazole compounds in agriculture may result in the occurrence of triazole resistant A. fumigatus isolates in the environment, not only by induction or selection of mutations in the cyp51A gene, but also by contribution to changes in the gene expression.
RESUMEN
We studied the presence of triazole resistance of 121 Aspergillus fumigatus clinical isolates collected in two Polish cities, Warsaw and Wroclaw, to determine if resistance is emerging in our country. We identified five itraconazole resistant isolates (4.13%) carrying the TR34/L98H alteration in Cyp51A gene, four of which were cross-resistant to posaconazole and one to voriconazole. One isolate was intermediate susceptible to itraconazole and harbored no Cyp51A alterations. The study confirms the presence of azole resistant A. fumigatus strains in Poland at a level that is comparative to other European countries.
Asunto(s)
Antifúngicos/farmacología , Aspergilosis/microbiología , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/aislamiento & purificación , Farmacorresistencia Fúngica , Triazoles/farmacología , Ciudades , Sistema Enzimático del Citocromo P-450/genética , Proteínas Fúngicas/genética , Genotipo , Humanos , Mutación Missense , PoloniaRESUMEN
The aim of this study was to evaluate the accuracy of commercial gradient test (Etest) in the detection of triazole resistant Aspergillus fumigatus isolates using reference microdilution methods and the analysis of sequences of the cyp 51A gene. The study was performed on twenty clinical isolates which were identified as Aspergillus fumigatus based on the DNA sequences of the ITS1-2 fragment of ribosomal DNA and the ß-tubulin gene, out of them seventeen isolates showed wild-type cyp51A sequence and three were positive for the mutation TR34/L98H. All isolates were tested for the susceptibility to itraconazole (ITZ), voriconazole (VOR) and posaconasole (POS) using microdilution methods, according to EUCAST and CLSI protocols, as well as using Etest. The results of microdilution and Etests were analysed separately according to clinical breakpoints (CBP) defined by EUCAST version 7.0 and epidemiological cut off values (ECV). Etest as well as reference methods excellently recognised the WT isolates, which were susceptible to all tested triazoles, regardless of the method and CBP or ECV criteria used. The Etest recognized three non-WT isolates as resistant or intermediately sensitive to ITZ and POS and one as resistant to VOR. The categorical concordance between Etests and EUCAST and Etests and the CLSI method ranged from 90 to 100%. The interpretation of the results obtained from routine A. fumigatus Etests requires great caution. The use of the confirmative examinations with reference AST methods as well as with molecular tests is recommended.
Asunto(s)
Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/genética , Pruebas Antimicrobianas de Difusión por Disco/métodos , Farmacorresistencia Fúngica/efectos de los fármacos , Proteínas Fúngicas/genética , Aspergillus fumigatus/genética , Farmacorresistencia Fúngica/genética , Itraconazol/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Polimorfismo Genético , Triazoles/farmacología , Voriconazol/farmacologíaRESUMEN
Aspergillus fumigatus is one of the most prevalent airborne fungal pathogens causing infections worldwide. Most A. fumigatus strains are susceptible to azoles, which are administered as the first line therapeutics. However, during last decade the acquired resistance to triazoles by these species has been described. There is a number of publications concerning the examination of clinical A. fumigatus strains from different countries, however there has been no report from Poland. Here, we describe for the first time, an examination of cyp51A and cyp51B expression level of 11 clinical A. fumigatus strains isolated during 2007-2014 period from the collection of Medical University in Wroclaw. Their susceptibility to itraconazole, voriconazole and posaconazole has been examined. The MIC values of triazoles for one of the examined isolates were respectively: > 8 mg/L for itraconazole, 2 mg/L for voriconazole and 0.5 mg/L for posaconazole. The cyp51A gene with its promoter region of all isolates was sequenced. It was found that the resistant isolate harbors the TR34/L98H mutation in the cyp51A gene and when cultured on media supplemented with voriconazole exhibits overexpression of both, cyp51A and cyp51B genes. The level of cyp51A gene expression was about 50 times higher than cyp51B.
Asunto(s)
Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Citocromos/genética , Voriconazol/farmacología , Aspergillus fumigatus/genética , Aspergillus fumigatus/aislamiento & purificación , Farmacorresistencia Microbiana , PoloniaRESUMEN
Development of effective and safe therapeutic treatment of fungal infections remains one of the major challenge for modern medicine. The aim of presented investigation was to analyze the in vitro antifungal activity of selected essential oils, ethanolic extracts of propolis and silver nanoparticles dropped on TiO2 against azole-resistant C. albicans (n = 20), C. glabrata (n = 14) and C. krusei (n = 10) clinical isolates. Among tested essential oils, the highest activity has definitely been found in the case of the oil isolated from the bark of Cinnamomum cassia, with MIC and MFC values for all tested strains in the range of 0.0006-0.0097 % (v/v) and 0.0012-0.019 % (v/v), respectively. High activity was also observed for the Lemon, Basil, Thyme, Geranium and Clove (from buds) essential oils. Significant differences in fungicidal activity have been observed in the case of four tested propolis samples. Only one of them revealed high activity, with MFC values in the range from 0.156 to 1.25 % (v/v). Satisfactory fungicidal activity, against C. albicans and C. glabrata isolates, was also observed in the case of silver nanoparticles, however C. krusei isolates were mostly resistant. We also revealed that constituents of most of essential oils and propolis as well as silver nanoparticles are not substrates for drug transporters, which belong to the most important factors affecting resistance of Candida spp. clinical isolates to many of conventional antimycotics. To conclude, the results of our investigation revealed that essential oils, propolis and silver nanoparticles represent high potential for controlling and prevention candidiasis.